Members of the erythroid Krü ppel-like factor (EKLF) multigene family contain three C-terminal zinc fingers, and they are typically expressed in a limited number of tissues. EKLF, the founding member, transactivates the ␤-globin promoter by binding to the CACCC motif. EKLF is essential for expression of the ␤-globin gene as demonstrated by gene deletion experiments in mice. Using a DNA probe from the zinc finger region of EKLF, we cloned a cDNA encoding a member of this family from a human vascular endothelial cell cDNA library. Sequence analysis indicated that our clone, hEZF, is the human homologue of the recently reported mouse EZF and GKLF. hEZF is a single-copy gene that maps to chromosome 9q31. By gel mobility shift analysis, purified recombinant hEZF protein bound specifically to a probe containing the CACCC core sequence. In co-transfection experiments, we found that sense but not antisense hEZF decreased the activity of a reporter plasmid containing the CACCC sequence upstream of the thymidine kinase promoter by 6-fold. In contrast, EKLF increased the activity of the reporter plasmid by 3-fold. By fusing hEZF to the DNA-binding domain of GAL4, we mapped a repression domain in hEZF to amino acids 181-388. We also found that amino acids 91-117 of hEZF confer an activation function on the GAL4 DNA-binding domain.

Endotoxic shock is a life-threatening consequence of severe Gram-negative infection characterized by vascular smooth muscle cell relaxation and severe hypotension. The production of nitric oxide (NO), through the inducible NO synthase pathway, has been implicated as a major contributor in this process. We now demonstrate that heme oxygenase (HO), an enzyme that generates carbon monoxide (CO) in the course of heme metabolism, may also be involved in the hemodynamic compromise of endotoxic shock. Inducible HO (HO-1) mRNA levels are dramatically increased in aortic tissue from rats receiving endotoxin, and this increase in vascular HO-1 message is associated with an 8.9-fold increase in HO enzyme activity in vivo. Immunocytochemical staining localizes an increase in HO-1 protein within smooth muscle cells of both large (aorta) and small (arterioles) blood vessels. Furthermore, zinc protoporphyrin IX, an inhibitor of HO activity, abrogates endotoxin-induced hypotension in rats. Studies performed in rat vascular smooth muscle cells in vitro show that the induction of HO-1 mRNA is regulated at the level of gene transcription, and this induction is independent of NO production. Taken together, these studies suggest that the up-regulation of HO-1, and the subsequent production of CO, contributes to the reduction in vascular tone during endotoxic shock.Endotoxemia leading to shock is a detrimental consequence of severe Gram-negative bacterial infection. Endotoxic shock is initiated by the release of bacterial cell wall-derived lipopolysaccharide (LPS) 1 and the subsequent production of cytokines and vasoactive mediators that result in vascular smooth muscle cell relaxation and hypotension (1, 2). One of the most important cytokines in the cascade of events leading to LPSinduced hypotension is interleukin (IL)-1␤ (1, 3). We have demonstrated previously that IL-1␤ stimulates the inducible isoform of nitric oxide synthase (NOS) and increases the production of NO in vascular smooth muscle cells (4). NO is a labile, free radical gas that acts as a potent vasodilator (5, 6). The importance of NO in the pathogenesis of endotoxic shock has been emphasized by recent studies demonstrating that mice carrying a disrupted inducible NOS gene have an attenuated hypotensive-response to LPS (7) and are resistant to LPS-induced death (7,8). However, the study by MacMicking and colleagues (7) also suggested that an inducible NOS-independent pathway contributes to LPS-induced hypotension and death, and we hypothesize that one potential pathway involves heme oxygenase (HO).HO is the enzyme that generates carbon monoxide (CO) and biliverdin (subsequently reduced to bilirubin) in the course of heme metabolism (9). CO is a gas molecule that shares some of the properties of NO, inasmuch as CO binds to the heme moiety of cytosolic guanylyl cyclase to produce cGMP (10). Two distinct forms of heme oxygenase have been identified (9): HO-1 (an inducible isozyme) and HO-2 (a non-inducible isozyme). Morita and colleagues (11) have demonstrated that HO-...

Several members of the LIM protein family have important roles in development and differentiation. We recently isolated a rat cDNA encoding a new member of this family, CRP2/SmLIM, that contains two LIM domains and is expressed preferentially in vascular smooth muscle cells (VSMC). To study the molecular mechanisms that regulate VSMC-specific transcription of the Crp2/SmLim gene, we cloned the cDNA and gene of mouse Crp2/SmLim. Mouse Crp2/SmLim is a single copy gene of six exons and five introns spanning approximately 20 kilobases of genomic DNA. By 5-rapid amplification of cDNA ends and S1 nuclease protection assay, we determined that the transcription start site is an A residue 80 base pairs 5 of the translation initiation codon. A TATA-like sequence is located 27 base pairs 5 of the transcription start site, and there are potential cis-acting elements (GATA, Sp1, AP-2, E box, CCAC box, and GArC motif) in the 5-flanking sequence. In transient transfection assays in rat aortic smooth muscle cells in primary culture, 5 kilobases of the Crp2/SmLim 5-flanking sequence generated a high level of luciferase reporter gene activity. By deletion analysis and gel mobility shift assay, we found that the region between bases ؊74 and ؊39 of this 5 kilobase DNA fragment binds Sp1 and confers basal promoter activity in the Crp2/ SmLim gene. In vitro, the 5-kilobase fragment was active in multiple cell types. In vivo, however, the 5-kilobase fragment directed high level expression of the lacZ reporter gene preferentially in the VSMC of transgenic mice, indicating the presence of VSMC-specific element(s) in this fragment.